Long-chain carboxychromanols and analogs for use as anti-inflammatory agents

ABSTRACT

Long-chain carboxychromanol compounds useful for treating conditions associated with the need to inhibit cyclooxygenase-1, cyclooxygenase-2, and/or 5-lipoxygenase, and pharmaceutical formulations containing the compounds are provided herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/119,737, filed Mar. 18, 2011, which is a U.S. nationalapplication under 35 U.S.C. §371(b) of International Patent ApplicationNo. PCT/US2009/057293, filed Sep. 17, 2009, which claims the benefitunder 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/098,357,filed Sep. 19, 2008, the entirety of the disclosures of which areincorporated herein by reference.

GOVERNMENT RIGHTS STATEMENT

This invention was made with government support under R01 AT001821, R21CA152588, & R01 AT006882 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND

The immune system plays a central role in maintaining health and diseasedevelopment. Excessive immune response leads to inflammation, which ischaracterized by the over-production of pro-inflammatory mediators,including lipid mediators, notably prostaglandins and leukotrienes, andcytokines like TNF-alpha, which in turn aggravate inflammation and leadto excessive damage to host tissues. During inflammation, several lipidmediators, such as prostaglandins and leukotrienes, arc synthesized fromthe essential fatty acid, arachidonic acid (AA), and play importantroles in mediating inflammatory response. For instance, prostaglandin E₂(PGE₂), which is synthesized from cyclooxygenase (COX)-catalyzedoxidation of AA, is believed to cause pain and fever as well as activatecytokine formation (44). Leukotriene B4, another oxidized productderived from AA through the 5-lipoxygenase (5-LO) catalyzed pathway inneutrophils, is a potent chemotactic agent. Important enzymes forprostaglandin formation are cyclooxygenases, which comprise aconstitutive form, COX-1, and an inducible form, COX-2. COX-1 catalyzedTxA2 formation in platelets activates platelet aggregation. Theprotective effect of low-dose aspirin in cardiovascular disease has beenattributed to its inhibition of COX-1-mediated TxA2 generation inplatelets. COX-2 is normally expressed in limited tissues but is inducedby endotoxin and cytokines in many immune cells including macrophages,monocytes and epithelial cells (45). Under most inflammatory conditions,COX-2 is up-regulated and is the primary enzyme responsible for theformation of pro-inflammatory PGE₂. 5-LOX has also been shown to play animportant role in inflammatory conditions including experimentalcolitis.

In addition to the lipid mediators, cytokines also play important rolesin regulating inflammatory response. The major pro-inflammatorycytokines, TNF-alpha and Interleukin 1-beta (IL-1beta), are known toactivate many immune cells such as monocytes and macrophages. Antibodiesagainst TNF-alpha and IL-1beta are clinically useful in the therapy ofcertain inflammatory diseases (49, 50).

These pro-inflammatory mediators are also believed to be important inthe development of degenerative diseases. For instance, various animaland human tumor tissues have been reported to express the enhanced COX-2and 5-LOX, as well as their products, PGE₂ and 5-HETE. PGE₂ has beenshown to promote proliferation of certain cancer cells, and NSAIDs caninhibit the growth of carcinoma cells and suppress angiogenesis. Inaddition to cancer, COX-2 and 5-LOX mediated reactions appear to play arole in cardiovascular diseases. Because of the central roles of PGE₂and LTB₄ in inflammation, COXs and 5-LOX have been recognized as targetsfor drug therapy in inflammatory diseases.

Although drugs targeting COXs have been extensively developed and usedin the treatment of inflammatory diseases, they are limited by adverseeffects Inhibition of both COX-1 and COX-2 by NSAIDs and selective COX-2inhibitors reduces the levels of prostaglandins, which leads to areduction of pain and inflammation. However, a selective shutdown ofCOXs pathway can cause alternative metabolism of arachidonic acid viathe 5-LOX pathway, which results in an increased production ofleukotrienes, such as LTB4 and cysteinyl leukotrienes. Theseleukotrienes are pro-inflammatory and also known to promotegastrotoxicity. Because of the disadvantage of the selective inhibitionof specific COXs pathways, a drug targeting COXs and 5-LOX, which canreduce both prostaglandins and leukotrienes, would provide a superioroutcome Inhibition of these multiple pathways can not only result in amore potent anti-inflammatory effect, but also reduce potential adverseeffect caused by a shunt in arachidonate metabolism to either pathway.

SUMMARY

The following presents a simplified summary of one or more embodimentsof the invention in order to provide a basic understanding of suchembodiments. This summary is not an extensive overview of allcontemplated embodiments, and is intended to neither identify key orcritical elements of all embodiments, nor delineate the scope of any orall embodiments. Its sole purpose is to present some concepts of one ormore embodiments in a simplified form as a prelude to the more detaileddescription that is presented later.

Inflammatory diseases affect millions of people in the world and chronicinflammation contributes to the development of degenerative diseasessuch as cancers, cardiovascular diseases, and neurodegenerativedisorders (1-3). Cyclooxygenases (COX) catalyze enzymatic oxidation ofarachidonic acid (AA) to prostaglandin H2 (PGH2), the common precursorto prostaglandins and thromboxane, which are important lipid mediatorsfor regulation of inflammatory response and other physiological as wellas pathophysiological processes (4, 5). Two COX isoforms have beenidentified. COX-1 is a constitutive form that regulates homeostasis inmainly tissues, and COX-2 is an inducible form that is mainlyresponsible for the generation of pro-inflammatory eicosanoids,including prostaglandin E₂ (PGE₂) under acute inflammatory conditions(5). COX inhibitors, which belong to non-steroidal anti-inflammatorydrugs (NSAIDs), have been used for the relief of fever, pain andinflammation (7, 8), as well as treatment for chronic diseases. Thisdisclosure provides conjugated long-chain carboxychromanol compoundsuseful for treating conditions associated with the need to inhibitcyclooxygenase-1, cyclooxygenase-2, and/or 5-lipoxygenase, such asdisorders associated with inflammation of the gut or various types ofcancer, and pharmaceutical formulations containing the compounds.

Other aspects and features, as recited by the claims, will becomeapparent to those skilled in the art upon review of the followingnon-limited detailed description of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described embodiments in general terms, reference will nowbe made to the accompanying drawings, which are not necessarily draw toscale.

FIG. 1A illustrates that vitamin E forms differentially inhibited PGE2in IL-1β treated A549 cells and the presence of sesamin partiallydecreased the inhibitory potency. A549 cells were pre-incubated withdifferent concentrations of tocopherols in the presence or absence of 1μM sesamin for 15 h, and then treated with IL-1β (2 ng/mL) for 24 h.PGE₂ in the cell-culture media was measured by ELISA assays. Results arethe averages of three independent experiments and expressed as Mean±SEM.

FIG. 1B illustrates that vitamin E forms differentially inhibited PGE2in IL-1β treated A549 cells and the presence of sesamin partiallydecreased the inhibitory potency. A549 cells were pre-incubated withdifferent concentrations of tocotrienols in the presence or absence of 1μM sesamin for 15 h, and then treated with IL-1β (2 ng/mL) for 24 h.PGE₂ in the cell-culture media was measured by ELISA assays. Results arethe averages of three independent experiments and expressed as Mean±SEM.

FIG. 1C illustrates that vitamin E forms did not significantly affectCOX-2 induction in 1L-1β activated A549 cells. The Western blot showedthe effect of vitamin E forms on COX.-2 induction. Cells are treatedwith vehicle (lane 1); 1L-1β (2 ng/ml, lane 2); or 1L-1β and γ-T at 40μM (lane 3), or δ-T at 40 μM (lane 4), or α-TE at 10 μM (lane 5), orγ-TE at 10 μM (line 6) for 24-h.

FIG. 2A illustrates dose-dependent accumulation of metabolites of δ-T incultured media. A549 cells were incubated with δ-T at 10, 25 and 50 μMfor 48 h. Media were collected and the metabolites were extracted andmeasured by HPLC.

FIG. 2B illustrates dose-dependent accumulation of metabolites of γ-T incultured media. A549 cells were incubated with γ-T at 10, 25 and 50 μMfor 48 h. Media were collected and the metabolites were extracted andmeasured by HPLC.

FIG. 2C illustrates that conditioned media showed dose-dependentinhibition of COX activity as assayed in intact cells. A549 cells wereactivated by 1L-1β (0.1 ng/mL) for 6 h to induce COX-2. Cells withpre-induced COX-2 were then incubated with “metabolites-containingmedium” obtained from the experiments disclosed in FIGS. 2A and 2B for30 min, and then added with AA (5 μM) and incubated for 5 min. Mediawere collected to measure PGE₂ formation. The relative COX activity wasexpressed as the ratio of PGE₂ under each treatment to that of vehiclecontrol media which were obtained under the same condition asmetabolite-conditioned media. All the results are averages of three ormore independent experiments (Mean±SD).

FIG. 3A illustrates that unconjugated long-chain carboxychromanols butnot sulfated derivatives inhibited COX-2 activity in intact cells. FIG.3A showed time-dependent changes of carboxychromanols and sulfatedcarboxychromanols in A549 cells. Sulfated metabolites were the sum of9′S, 11′S and 13′S, and unconjugated metabolites are the sum of 9′, 11′and 13′. Conditioned media were obtained by incubation of A549 cellswith γ-TE at 20 μM for 24, 48 and 72 h. Metabolites were extracted andmeasured using HPLC assay. The conditioned media were then used for theactivity assay as described in FIGS. 2A and 2B. Unsulfated/sulfated isthe ratio of the sum of 9′, 11′ and 13′ to that of 9′S, 11′S and 13′S.All the results are expressed as Mean±SD.

FIG. 3B illustrates the inhibitory potency correlated with theaccumulation of unconjugated long-chain carboxychromanols but not thatof sulfated forms. Conditioned media were obtained by incubation of A549cells with γ-TE at 20 μM for 24, 48 and 72 h. Metabolites were extractedand measured using HPLC assay. The conditioned media were then used forthe activity assay as described in FIGS. 2A and 2B. Unsulfated/sulfatedis the ratio of the sum of 9′, 11′ and 13′ to that of 9'S, 11'S and13'S. All the results are expressed as Mean±SD.

FIG. 4. δTE-13′ inhibits cancer cell growth and induces cancer celldeath. Panel A shows dose-dependent inhibition of human breast MCF-7cell growth based on different concentrations of δTE-13′ compared to acontrol, as indicated by MTT assays. Panel B shows dose-dependentinhibition of colon HCT116 cancer cell growth based on differentconcentrations of δTE-13′ compared to a control, as indicated by MTTassays. Panel C shows that δTE-13′ effectively kills cancer cells byinduction of apoptosis in HCT116 colon cancer cells as indicated by flowcytometry data.

FIG. 5. Effects of δTE-13′ on AOM-DSS-induced colon inflammation andtumorigenesis. Panel A shows the study design having δTE-13′ at 0.025%(w/w) in AIN93G based diet. Panel B shows a fecal bleeding score on the7th day of the 2nd cycle of DSS. Panel C shows the effects of δTE-13′ ontumor multiplicity. **P<0.01, *P<0.05: significant difference betweenδTE-13′ and control group (n=15-16).

FIG. 6. Detection of δTE-13′-1 in fecal samples. The panel shows aliquid chromatography-mass spectrometry analysis of fecal samples ofmice fed with δTE-13′.

FIG. 7. δTE-13′ ameliorated colitis induced by DSS in mice. Panel Ashows the study design including when DSS and δTE-13′ were introducedand when tissue was harvested. Panel B shows the significant differentin DSS-induced diarrhea and fecal bleeding in control mice compared totreatment mice.

DETAILED DESCRIPTION

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all, embodiments are shown. Indeed, the disclosure may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Additionally, while embodiments are disclosed as“comprising” elements, it should be understood that the embodiments mayalso “consist of” elements or “consist essentially of” elements. Wherepossible, any terms expressed in the singular form herein are meant toalso include the plural form and vice versa unless explicitly statedotherwise. Also, as used herein, the term “a” and/or “an” shall mean“one or more,” even though the phrase “one or more” is also used herein.Like numbers refer to like elements throughout.

Throughout this disclosure, various information sources are referred toand/or are specifically incorporated. The information sources includescientific journal articles, patent documents, textbooks, and websites.While the reference to these information sources clearly indicates thatthey can be used by one of skill in the art, each and every one of theinformation sources cited herein are specifically incorporated in theirentirety, whether or not a specific mention of “incorporation byreference” is noted. The contents and teachings of each and every one ofthe information sources can be relied on and used to make and useembodiments of the disclosure.

The disclosure provides long-chain carboxychromanol compounds useful fortreating conditions associated with the need to inhibitcyclooxygenase-1, cyclooxygenase-2, and/or 5-lipoxygenase, andpharmaceutical formulations containing the compounds.

DEFINITIONS

The term “carrier” is used herein to describe any ingredient other thanthe active components in a formulation. The choice of carrier will to alarge extent depend on factors such as the particular mode ofadministration, the effect of the carrier on solubility and stability,and the nature of the dosage form.

The term “patient” refers to mammals, including humans, companionanimals, and livestock animals.

“Pharmaceutically acceptable” as used in this application, for examplewith reference to salts, polyphenolic sulfation inhibitors, andformulation components such as carriers, means substantiallynon-deleterious to the recipient patient, and includes “veterinarilyacceptable,” and thus includes both human and animal applicationsindependently.

The term “polyphenolic sulfation inhibitor” includes those compoundswhich can inhibit the long-chain carboxychromanol compounds frommetabolizing or converting in whole or in part to a sulfated form of thecompound. Such pharmaceutically acceptable polyphenolic sulfationinhibitors include, for example, sesamin and curcumin.

The term “therapeutic amount” means an amount of a compound sufficientto treat one or more physiological disorders associated an excess ofCOX-1, COX-2, and/or 5-LOX. The specific dose administered is determinedby the particular circumstances surrounding each patient's situation.These circumstances include the route of administration, the priormedical history of the patient, the particular physiological disorder orsymptom being treated, the severity of the particular physiologicaldisorder or symptom being treated, and the age and sex of the patient.However, it will be understood that the therapeutic dosage administeredwill be determined by a physician in light of the relevantcircumstances, or by a veterinarian for non-human patents. Generally, adosage amount of between about 0.01 to 1000 mg/kg of weight of thepatient can be employed, and administered once or more daily, weekly, ormonthly, depending on the circumstances described above.

The terms “treat”, “treating”, and “treatment” include ameliorating,halting, slowing, restraining, and reversing the progression of orreducing the severity of the physiological disorders, or their symptoms,associated with the need to inhibit COX-1, COX-2, and/or 5-LOX.

Long-Chain Carboxychromanol Compounds and Method of Using Same

The long-chain carboxychromanol compounds inhibited COX-1, COX-2, and5-LOX. As such the compounds are of value in the treatment of a widevariety of clinical conditions which are characterized by the presenceof an excess of COX-1, COX-2, and/or 5-LOX. Thus, the invention providesmethods for the treatment or prevention of a physiological disorderassociated with an excess of COX-1, COX-2, and/or 5-LOX, which methodcomprises administering to a mammal in need of said treatment aneffective amount of a long-chain carboxychromanol compound or apharmaceutically acceptable salt thereof. The terms “physiologicaldisorder associated with an excess of COX-1”, or “ . . . COX-2”, or “ .. . 5-LOX” encompass those disorders associated where inhibition ofCOX-1, COX-2, and/or 5-LOX is desired to alleviate the disorder and/orits symptoms. Such disorders include, for example, arthritis, rheumatoidarthritis, spondyloarthopathies, gouty arthritis, osteoarthritis,systemic lupus erythematosus, juvenile arthritis, gastrointestinalconditions (e.g., inflammatory bowel disease, Crohn's disease,gastritis, irritable bowel syndrome, ulcerative colitis, and the like),colorectal and other cancers, asthma, bronchitis, menstrual cramps,tendinitis, bursitis, skin related conditions (such as, for example,psoriasis, eczema, burns, dermatitis, and the like), vascular diseases,periarteritis nodosa, thyroidiris, aplastic anemia, Hodgkin's disease,sclerodoma, rheumatic fever, type I diabetes, myasthenia gravis,sarcoidosis, nephrotic syndrome, Behcet's syndrome, potymyositis,gingivitis, hypersensitivity, conjunctivitis, swelling occurring afterinjury, myocardial ischemia, and the like, as well as others mentionedelsewhere herein.

Pharmaceutically acceptable salts and common methodology for preparingthem are known in the art. See, e.g., P. Stahl, et al., Handbook ofPharmaceutical Salts: Properties, Selection And Use, (VCHA/Wiley-VCH,2002); S. M. Berge, et al., “Pharmaceutical Salts,” Journal ofPharmaceutical Sciences, Vol. 66, No. 1, January 1977. Examples of saltsinclude, but are not limited to, salts formed by standard reactions withboth organic and inorganic acids, such as sulfuric, hydrochloric,phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, cholic,pamoic, mucic, glutamic, camphoric, glutaric, glycolic, phthalic,tartaric, formic, lauric, stearic, salicylic, methanesulfonic,benzenesulfonic, sorbic, picric, benzoic, cinnamic and like acids.

The compounds of the present invention are preferably formulated aspharmaceutical compositions administered by a variety of routesincluding the oral, rectal, transdermal, subcutaneous, topical,intravenous, intramuscular or intranasal routes. Such pharmaceuticalcompositions and processes for preparing same are well known in the art.See, e.g., Remington: The Science and practice of pharmacy (A. Gennaro,et al., eds., 19th ed., Mack Publishing Co, 1995).

In one embodiment, long-chain carboxychromanols and related compoundsuseful in the invention are of the following formula:

where X is 0, CH₂, or NH;Y is OH, NH, —O(C₁-C₆ alkyl), or —OC(O)O(C₁-C₆ alkyl);R₁ is H or C₁-C₆ alkyl;R₂ is H or C₁-C₆ alkyl;R₃ is H or C₁-C₆ alkyl;R₄ is C₉-C₁₇ straight chain alkyl, optionally substituted by one or moreC₁-C₆ alkyl, and having a carboxy group (—COOH) at its terminal end; andpharmaceutically acceptable salts thereof “C₁-C₆ alkyl” includes thosebranched or straight chain substituents having 1 to 6 carbons andincludes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl,isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl,isohexyl, and the like. “C₉-C₁₇ straight chain alkyl” includes thosestraight chain substituent's having from 9 to 17 in the chain such asnonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,hexadecyl, and heptadecyl, and which may be further substituted with oneor more of C₁-C₆ alkyl. Scheme 1 further illustrates the compoundsuseful in the invention.

Inflammatory diseases affect millions of people in the world and chronicinflammation contributes to the development of degenerative diseasessuch as cancers, cardiovascular diseases, and neurodegenerativedisorders (1-3). Cyclooxygenases (COX) catalyze enzymatic oxidation ofarachidonic acid (AA) to prostaglandin H2 (PGH2), the common precursorto prostaglandins and thromboxane, which are important lipid mediatorsfor regulation of inflammatory response and other physiological as wellas pathophysiological processes (4, 5). Two COX isoforms have beenidentified. COX-1 is a constitutive form that regulates homeostasis inmainly tissues, and COX-2 is an inducible form that is mainlyresponsible for the generation of pro-inflammatory eicosanoids,including prostaglandin E2 (PGE₂) under acute inflammatory conditions(5). COX inhibitors, which belong to non-steroidal anti-inflammatorydrugs (NSAIDs), have been used for the relief of fever, pain andinflammation (7, 8), as well as treatment for chronic diseases. It isnow well established that NSAIDs are effective and usefulchemoprevention agents for colon cancer (9) and possibly other types ofcancer (10).

Vitamin E comprises four tocopherols (α-, β-, γ-, and δ-T) and fourtocotrienols (α-, (β-, γ-, and δ-TE)(Scheme 2). α-T is the predominantvitamin E form in the plasma and tissues, as well as in mostsupplements. γ-T, primarily found in plant seeds and plant oils, is themajor vitamin E form in the US diets (11). γ-T and δ-T togetherconstitute 70-80% of vitamin E in the US diet. Rich sources oftocotrienols include palm oil, cereal and barley (11). Until recently,α-T was the only vitamin E form had drawn most attention and extensivelystudied. Recent studies by us and others indicate that other forms ofvitamin E have distinct bioactivities from α-T, and these properties maybe important to disease prevention and/or therapy (12). Specifically, wehave showed that γ-T and its terminal metabolite, γ-CEHC([(2-carboxyethyl)-hydroxychroman]), inhibited COX-2 catalyzed PGE₂formation in LPS activated macrophage and 1L-1β-treated epithelial cells(13). In contrast, α-T was much less effective. In a rat inflammationmodel, we showed that γ-T and γ-CEHC inhibited proinflammatoryeicosanoid formation and attenuated inflammation-induced damage (14). Wealso documented that γ-T, in contrast to α-T, inhibited growth andinduced death in cancer cells but had no effect on normal epithelialcells (15).

Recently, we and others have shown that vitamin E forms are metabolizedto long-chain carboxychromanols, i.e. 9′-, 11′-, 13′-carboxychromanol(16-18) and their sulfated counterparts (17) (Scheme 3).

These metabolites are generated by w-hydroxylation and oxidation of theco-terminal carbon to generate 13′-carboxychromanol, followed by astep-wise 13-oxidation to remove 2- to 3-carbon moiety each cycle toform shorter side-chain carboxychromanols. The terminal urinary-excretedmetabolite is CEHC (3′-carboxychromanol) (16, 19). During this process,significant amounts of sulfated long-chain carboxychromanols are alsogenerated (17). Importantly, we showed that some of these metaboliteswere found in rat plasma subsequent to supplementation (17).

In the present study, we systemically examined the effect of differentvitamin E forms and their metabolites on COX-2 catalyzed PGE₂ formationin IL-10 activated human lung A549 cells, as well as the effect on COXactivity in enzyme assays. We found that 13′-carboxychromanol is apotent inhibitor of COXs, and carboxychromanols with shorter side chainincluding 9′ and CEHC, as well as vitamin E are weaker inhibitors. Onthe other hand, the sulfated derivatives appeared to be ineffective.

Materials—

αT (99%), γT (97%,99%), and δT (97%) were purchased from Sigma

(St Louis, Mo.). γ-CEHC (>98%) was from Cayman Chemicals (Ann Arbor,Mich.). α-Tocotrienol (α-TE) and γ-tocotrienol (γ-TE) were a generousgift from BASF (Germany). Tissue culture reagents were from Invitrogen(Rockville, Md.). Monoclonal COX-2 antibody, human recombinant COX-2 andovine COX-1 were obtained from Cayman Chemicals (Ann Arbor, Mich.).Human recombinant interleukin-1 (IL-1β), sesamin, ketoconazole, dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT), and all other chemicals were from Sigma.

Cell Culture—

Human lung A549 cancer cells were obtained from American Type CultureCollection (Manassas, Va.). These cells were routinely cultured inRPMI-1640 with 10% fetal bovine serum (FBS).

PGE₂ Generation During Chronic IL-1β Treatment—

2.5-3×10⁵ A549 cells per well were seeded in RPMI-1640 with 10% of FBSand allowed to attach overnight in a 24-well plate. Vitamin E stocksolutions were initially made in DMSO and then diluted in 10 mg/mL ofbovine serum albumin. Confluent cells were incubated in DMEM containing1% FBS with DMSO (control) or vitamin E forms for 14-16 h and then 2ng/ml of IL-1β was introduced for 24 hours. The medium was thencollected and PGE₂ accumulation was measured using ELISA assay fromCayman Chemicals (Ann Arbor, Mich.).

COX-2 Activity in Intact Cells—

A549 cells were pre-treated with 0.5-1 ng/mL of IL-1β- for 6 hours toinduce COX-2 expression, then incubated with fresh medium containingvitamin E farms, metabolite-containing conditioned medium or controlmedium for 30 min. In some experiments, isolated 9′ and 13′, as well astheir controls, were added for the 30-min preincubation. The enzymereaction was initiated by addition of 5 μM AA for 5 min, and medium wascollected and immediately frozen to −20 C. PGE₂ generated was measuredas an index of COX-2 activity using an EIA assay from Cayman Chemicals.

COX-1 and COX-2 Activity Assay Using Purified Enzymes—

The enzymatic reactions were performed in 0.1M Tris (pH 8.0), in thepresence of 5 mM EDTA, 2 mM phenol, and 1 μM heme. Tested compounds,including ibuprofen, acetaminophen, isolated 9′-COOH or 13′-COON, werefirst preincubated with ovine COX-1 or human recombinant COX-2 for 10min at room temperature. Enzymatic reactions were initiated by additionof AA at a final concentration of 5 μM for 2 min. The reaction wasstopped by addition of 0.1 M HCl. Stannous chloride in 0.1 M HCl wasthen added to reduce PGG₂ and PGH₂ to PGF_(2a), After addition of 0.5vol of 03M NaCl, prostaglandins formed in the reaction were extractedusing 2.5 vol of ethyl acetate and the organic layer was completelydried under N₂. PGF_(2a), and PGE₂ were quantified using ELISA assaysfrom Cayman Chemicals. Under these experimental conditions, PGF_(2a) isthe predominant product.

Cellular Uptake of Vitamin E Forms—

Cells were incubated in DMEM containing 1 FBS supplemented withdifferent vitamin E forms for 24 hours. After harvested by scraping,cells were washed twice with HBSS. Cellular uptake of vitamin E was thenquantified using an HPLC with EC detection (13).

Conditioned Medium Containing Long-Drain Vitamin E Metabolites—

A549 cells were seeded in RPMI-1540 with 10% FBS at a density of 8×10⁵cells per well in 6-well plates. Twenty-four hours later, media werereplaced with fresh DMEM containing 1% FBS with vitamin E forms, or DMSD(0.05%) in controls. Cells were incubated for 24-72 h as specified inthe results. Metabolite-containing media were collected, frozenimmediately and stored in −20° C. until use.

Quantitation of Vitamin E Metabolites in Conditioned Media—

Long-chain carboxychromanols and their sulfated counterparts werequantitated by a HPLC assay with fluorescent detection (17). Briefly, 8μL of ascorbic acid (11 mg/mL) was added to 400 μL of conditionedmedium, which was then mixed with 10 μL of ethanol and 500 μL of hexane.The mixture was vortexed for 1 min, and followed by centrifugation at13000 rpm for 2 min. The hexane layer was discarded and the aqueousphase was acidified using 14 μL of acetic acid. The aqueous phase wasextracted twice with 1 mL of ethyl acetate, vortexed and centrifuged.The combined ethyl acetate layers were dried under nitrogen. The residuewas reconstituted in 200 μl of 70% MeOH/30% water and injected onto theHPLC column.

Extracted metabolites were separated using HPLC and detected by aShimadzu RF-10AXL spectrofluorometric detector (Shimadzu, Columbia, Md.)with the excitation and emission wavelength at 292 nm and 327 nm,respectively. The mobile phases included A—35% acetonitrile, 65% 10 mMammonium acetate at pH 4.3 and B—96% acetonitrile, 4% 10 mM ammoniumacetate at pH 4.3. The metabolites were separated on a 5 micronSupelcosil LC-18-DB column, 4.6×150 mm (Supelco, Bellefonte, Pa.) usinga flow rate of 1.0 mL/min with the following gradient: maintaining 100%A for 8 min, linearly increasing to 100% B from 8 to 30 min, maintaining100% B until 55 min and then back to 100% A at 56 min. γ-CEHC wasquantified using the authentic standard as the external standard.Long-chain metabolites were quantified using tocopherols as the externalstandards with a correction factor based on the linear relationshipbetween fluorescent intensity and solvent content (17).

Western Blot—

Cells were lysed in Tris-EDTA, 1% SIDS, 1 mM DTT with protease inhibitorcocktails (Sigma) and the resulting solution was heated at 95° C. for 5min. Equal amounts of protein (10-25n) were loaded on 10-12% pre-castSDS-PAGE gels (BioRad, Richmont Calif.). Resolved proteins weretransferred onto a PVDF membrane (Millipore) and probed by antibodies.Membranes were exposed to chemiluminescent reagent (NEN, Life ScienceProducts) and visualized on a Kodak film using a M35A X-GMAT processor(Kodak).

Statistical Analysis—

The unpaired student's t-test was used in the statistical analysis. Allresults are expressed as mean±SD. Activation of human lung epithelialA549 cells by IL-1β leads to a strong up-regulation of COX-2 proteinexpression and almost 100-fold increase of PGE₂ generation. Thiscellular system has been employed to evaluate inhibitory potency ofanti-inflammatory drugs, including COX inhibitors and was previouslyused by us to study the effect of α-T, γ-T and γ-CEHC on PGE₂ formation(13, 20). In the present study, we found that various forms of vitamin Edifferentially inhibited prostaglandin E₂ (PGE₂) formation when A549cells were co-treated with IL-1β and vitamin E forms (FIG. 1). Comparedwith γ-T, δ-T and γ-TE appeared to be even stronger inhibitors, whereasα-T, β-T (no inhibition at 50 μM) and α-TE (20% inhibition at 20 μM) aremuch less effective at physiologically relevant concentrationsInhibition of PGE₂ by γ-T, and γ-TE was also observed in the presence ofexogenous AA, where after co-incubated with vitamin E forms and IL-1β,cells were incubated with fresh media containing 5 μM of AA for 5 min.Under this condition, the concentrations of γ-T, δ-T and γ-TE to cause50% inhibition increased to 25, 10 and 10 μM, respectively. Thissuggests that the inhibition was independent of substrate availability,and vitamin E forms appear to be stronger inhibitors in the presence ofendogenous AA. Using Western Blot, we found that co-incubation withvitamin E forms did not significantly affect the induction of COX-2protein in response to IL-1β activation (FIG. 1 C), which is consistentwith our previous observations (13). These results suggest that theinhibitory effect may stem from their inhibition of COX activity.

It has been demonstrated that γ-Y, δ-T, and γ-TE are metabolized in A549cells to form long-chain carboxychromanols, i.e., 9′, 11′, and 13′ (17,18) and sulfated carboxychromanols, 9′S, 11′S, and 13′S (17). Toinvestigate whether the metabolism of vitamin E forms affect theirinhibition of PGE₂, we used sesamin, which is an inhibitor of tocopherolω-hydroxylase (21) and almost completely inhibited the catabolism ofvitamin E forms (17). The presence of sesamin significantly reduced theinhibitory potency of γ-T, while sesamin alone, at 1 μM, did not affectPGE₂ generation (FIG. 1A). Sesamin also moderately diminished theinhibitory potency of δ-T and γ-TE (FIGS. 1 A and B). The similar effectwas observed with the presence of another cytochrome P-454 inhibitor,ketoconazole. These observations suggest that inhibition of PGE₂ is, inpart, attributed by the metabolites generated from vitamin E in thiscellular system.

We then examined whether vitamin E forms affect cell viability becauseprevious studies showed that γ-T and δ-T inhibited growth and inducedapoptosis in several cancer cell lines (15, 22). Under the currentexperimental conditions, where cells were 100% confluent and incubatedwith vitamin E in the presence of 1% FBS, γ-T at 25-50 μM, δ-T at 25-50μM and γ-TE at 20 μM, did not show significant effects on cellviability, as indicated by MTT assays and no apparent changes in cellmorphology during the period of entire incubation.

To investigate whether vitamin E metabolites directly inhibit COXactivity, we tested a potentially inhibitory effect of conditionedmedia, which were obtained by incubation of vitamin E forms with A549cells to generate long-chain carboxychromanols and sulfatedcarboxychromanols (17). Concentrations of carboxychromanols and sulfatedcarboxychromanols in conditioned media, as quantified by a sensitiveHPLC assay with fluorescent detection (17), appeared to increaseproportionally with the dose of added vitamin E forms (FIG. 2A). Whentested in intact-cell assays, these metabolite-containing media showeddose-dependent inhibition of COX-2 activity in the presence ofendogenous AA (FIG. 2B). Conditioned media from δ-T were slightly morepotent than those from γ-T, probably because of higher concentrations ofmetabolites (FIG. 2). Three control experiments were performed toconfirm that the inhibition was due to the metabolites rather than theprecursor vitamin or non-specific oxidation products. Specifically,media obtained by a co-incubation of vitamin E and sesamin, or from acell-free system failed to show any inhibitory effects. In addition,freshly γ-made vitamin E forms did not directly show inhibition underthe assay condition

(Materials and Methods) (Experimental Conditions in FIG. 2).

Our previous studies showed that the terminal metabolite of γ-T, γ-CEHC,inhibit COX-2 activity using the intact cell assays (13). Because nosignificant amount of γ-CEHC were found in A549 cells (17), we reasonedthat long-chain metabolites are responsible for the reduction of COX-2activity.

We next asked whether COX inhibition stems from non-conjugatedlong-chain carboxychromanols, or sulfated derivative, or both. We tookadvantage of the observation that

90% metabolites from γ-TE were unconjugated carboxychromanols during thefirst 24-h incubation, whereas more than 85% metabolites were sulfatedcarboxychromanols when media were obtained after 72-h incubation (FIG.3A). Using the conditioned media obtained after 24, 48 and 72 hincubation, we found that the inhibitory potency gradually diminishedwhen metabolites shifted from non-conjugated long-chaincarboxychromanols (at 24 h) to sulfated derivatives which becamepredominant at 72 h (FIG. 3B). In contrast, for metabolites from δ-Twhich had minimal formation of sulfated metabolites (FIG. 2, (17)), atime-dependent enhanced inhibitory potency was observed parallel to atime-dependent increase of non-conjugated metabolites. These findingsstrongly suggest that carboxychromanols but not their sulfatedmetabolites are mainly responsible for the observed inhibitory effect.

To directly examine the effect of long-chain metabolites on COXactivity, we purified and isolated 9′- and 13′-carboxychromanol (Scheme4) from δ-T-conditioned media, because of their relative abundance.

In the activity assay in intact cells, we found that both purifiedmetabolites potently inhibited COX-2 activity. On the other hand, thesame fraction isolated from control media at the same retention time onHPLC, did not show significant effect. The IC5Os for 9′ and13′-carboxychromanol as assayed in intact cells was approximately 5-10μM (Table 1). Under the same conditions, ibuprofen and acetaminophenalso inhibited COX-2 activity with IC5Os of 5 and 300 μM, respectively.

TABLE 1 Long-chain carboxychromanols are inhibitors of COX-1 and COX-2.The effect of purified carboxychromanols, 9′ and 13′, on COX activitywas assayed in intact cells and using purified enzymes, as described inMaterials and Methods. Results were obtained based on two or threeindependent experiments and expressed as mean + SD. COX-2 COX-1 COX-2IC50 (μM) In A549 cells  9′ 7 ± 2 Not inhibit* Not inhibit* 13′ 6 ± 2 5± 2 4 ± 2 γ-CEHC 30-70^(a) 300 ± 50  450 ± 50  Ibuprofen 5 ± 2 8 ± 2 5 ±1 Acetaminophen 300 ± 50  Not inhibit* Not inhibit* *9′ andacetaminophen at 20 and 250 μM, respectively, did not show any effect ofCOX-1 or COX-2 activity. ^(a)previously reported (Grammas, 2004 #41;Jiang, 2000 #2).

Potential inhibition of COX-1 or COX-2 was further examined inenzyme-based assays. We found that 13′-carboxychromanols inhibited COX-1and COX-2 activity with an apparent IC50 of 4-7 μM, which is similar tothat of ibuprofen (Table 1). On the other hand, 9′-carboxychromanols atthe maximum concentration of 20 μM did not inhibit either enzyme. Wewere not able to evaluate the inhibitory effect of 9′ at higherconcentrations because of its limited resources. As a comparison,acetaminophen did not significantly inhibit COX-1 or COX-2 at 250 μM inthis assay system (Table 1). F-CEHC showed inhibitory effect at higherIC5Os (300-500 μM). Vitamin E forms are not effective at 50 μM, thehighest concentration used.

To further understand the differential effect observed between 9′ and13′, we used computer simulation to test the relative binding affinity.The data showed although both 9′ and 13′ appear to fit in the substratebinding pocket of COX-2, 13′ can interact more favorably with theenzyme, compared with 9′. This is consistent with the results fromenzyme assays (Table 1).

Cyclooxygenase-catalyzed generation of proinflammatory eicosanoids playsimportant roles in regulation of inflammatory response and contributesto chronic diseases such as cancer. A major finding of the current studyis that long-chain carboxychromanols, which can be generated fromvitamin E forms via co- and P-oxidation of the phy-tyl chain in cellsand rats (17, 18), are potent inhibitors of cyclooxygenases (Table 1),On the other hand, the sulfated carboxychromanols, which can also bederived from vitamin E (17), appear to be ineffective (FIG. 3). Wedemonstrated that although both 9′ and 13′ inhibited COX-2 activity inintact cells, 13′ was a much more potent inhibitor of COX-1 and COX-2,as indicated by enzyme-based assays, where 13′ shoved inhibitory potencysimilar to ibuprofen, a commonly used NSAID (Table 1). Compared withlong-chain carboxychromanols, γ-CEHC and vitamin E forms, such as γ-T,δ-T and γ-TE but not α-T, β-T or α-TE, appeared to be relatively weakerinhibitors of COX-2. Our study therefore identified certain long-chaincarboxychromanols as novel COX inhibitors.

The observation that 13′ is a more potent inhibitor than vitamin Eforms, 9′ and 3′ (γ-CEHC) indicates that the conversion of 13′-carbon toa carboxylic acid, and the length of side chain are important factorsfor COX inhibition by these chromanol analogs. It is known that thecarboxylate group of A, forms ion pair or a hydrogen bond with theguanidinium group of a conserved arginine (Arg120), and Tyr355 (23, 24).The importance of these interactions is evident by the observation thatsite-directed mutagenesis of Arg120 renders the protein resistant toinhibition by carboxylic acid-containing NSAIDs or certain COX-2inhibitors (25), and increases the Km for AA binding (26,27).

It is conceivable that the carboxylate group in long-chaincarboxychromanols is likely to have similar interaction with theguanidinium group of Arg120 and Tyr355, whereas no such interaction canbe formed with vitamin E forms. Using computer simulation, we found thatboth 13′ and 9′ can form an extended L-shaped conformation to fit in thesubstrate binding pocket of COX-2, and appeared to be capable ofinteracting with Arg120 and Tyr355. And yet, 13′ appears to interactstronger than 9′ with other hydrophobic amino acids at thesubstrate-binding site of the enzyme. Similarly, the longer side chainof 9′ renders it stronger interaction with the enzyme than γ-CEHC. Inaddition, the current study showed that sulfated long-chaincarboxychromanols do not inhibit COX activity (FIG. 3). This may be dueto the strong polarity of the sulfate group which cannot interactfavorably with the majority of hydrophobic amino acids at the bindingsite.

In a further embodiment, 13′-carboxychomanol analogs with conjugation atthe phenolic group, i.e., δT-13′-R and δTE-13′-R, have been determinedto be useful for prevention and treatment of cancer and inflammatorydiseases. These conjugated 13′carboxychromanol analogs are shown inscheme 5, which depicts variants of scheme 1 with conjugation at thephenolic group for both tocopherols (A) and tocotrienols (B). Dualinhibition of COX and 5-LOX is an important anti-inflammatory andanticancer property for prevention and treatment of cancer andinflammatory diseases (9, 63-65). Dual inhibition of COX and 5-LOX hasadvantages over non-steroid anti-inflammatory drugs such as NSAIDS, COXinhibitors, etc., by inhibiting multiple pro-inflammatory pathways withreduced adverse effects (66). Because of this activity, δTE-13′ iseffective in the treatment and prevention of cancer and inflammatorydiseases.

The 13′-carboxychromanol analog may be conjugated to an acetyl groupforming an acetate, a methyl group forming a methoxy group, or ahydrogen ion forming a hydroxyl group, or the like. In some embodiments,an advantage of using conjugated compounds (δT-13′-R or δTE-13′-R)instead of unconjugated compounds (δT-13′ or δTE-13′) is that conjugatedcompounds are less sensitive to oxidation and have better shelf life butare equally effective against cancer. For example, an acetate conjugatecan be hydrolyzed to form unconjugated carboxychromanols in the body tobecome active. An example of hydrolysis of conjugated compounds has beendescribed with tocopheryl acetate (70, 71).

Specific examples of conjugated 13′-carboxychromanol analogs are shownin Scheme 6 below. For example,13-(6-hydroxy-2,8-dimethylchroman-2-yl)-2,6,10-trimethyltridecanoic acidis δT-13′ analog. Importantly, new research has indicated that δTE-13′analogs such as(2E,6E,10E)-13-(6-hydroxy-2,8-dimethylchroman-2-yl)-2,6,10-trimethyltrideca-2,6,10-trienoicacid are effective in dual inhibition of COX and 5-LOX, in inducingcancer cell death, in suppression of tumorigenesis in a colon cancermodel, and in mitigating experimental colitis in mice.

A recent study indicates that δTE-13′ as shown in Scheme 6 inhibits bothcyclooxygenase (COX) and 5-lipoxygenase (5-LOX), similar to δT-13′.Table 2 discloses the inhibitory effect of 13′-COOHs on COX-2 and 5-LOX.The enzyme assays with purified COX were conducted as described in ref(67). Effects on 5-LOX activity were assessed by the ferrousoxidation-xylenol orange assay (68, 69).

TABLE 2 Inhibitory effect of 13′-COOHs on COX-2 and 5-LOX. IC50 (μM)δT-13′ δTE-13′ COX-2 activity in intact cells 4 ± 2 4 ± 1  Humanrecombinant 5-LOX (9U) 4 ± 1 2 ± 0.5

As shown in table 2, the COX-2 activity in intact cells was inhibited ina similar manner by both δT-13′ and δTE-13′. Unexpectedly, δTE-13′exhibited an even greater effect on inhibition of human recombinant5-LOX activity compared to δT-13′.

Turning now to FIG. 4, additional research has identified that δTE-13′dose-dependently inhibits human breast MCF-7 cancer call growth (panelA) and colon HCT116 cancer cell growth (panel B), as indicated by MTTassays. As shown in FIG. 4A and FIG. 4B, concentrations of δTE-13′ at 5μM, 10 μM, and 20 μM were compared to a control for over 60 hours. Therelative cell viability as a percentage of the control was determinedfor control and for each concentration. As the concentration of δTE-13′increases, the trend in cell viability changes. For both cell types,when the concentration reaches 20 μM δTE-13′ the trend in relative cellviability is negative over time.

In FIG. 4C, flow cytometry data is presented that indicates that δTE-13′effectively kills cancer cells by induction of apoptosis in HCT116 coloncancer cells. In the flow cytometry data, the control indicates that thecancer cells were predominately healthy (lower left quadrant, 94.8%)with only small portions in early stage apoptosis (lower right quadrant,2.32%) or late stage apoptosis (upper right quadrant, 2.57%). Incontrast, cancer cells in the treatment group had a much largerproportion in early stage apoptosis (lower right quadrant, 7.92%) andlate stage apoptosis (upper right quadrant, 22.9%) when treated with 20μM δTE-13′ after 24 hours. Similar data were observed with δT-13′ (datanot shown).

In FIG. 5, the effect of δTE-13′ on tumorigenesis in azoxymethane(AOM)/dextran sulfate sodium (DSS)-induced colon cancer in Balb/c miceis investigated. AOM is a carcinogen and DSS administered in drinkingwater causes colon inflammation that promotes colon tumorigenesis.Therefore, the AOM-DSS model represents inflammation (colitis)-promotedcolon cancer. In FIG. 5A, the experimental design is shown. At day 0,AOM is administered to the Balb/c mice. 1.5% DSS in drinking water isintroduced and δTE-13′ (0.025% w/w) supplementation in AIN93G based dietbegins at day 7. Water is introduced at day 14, 1.5% DSS is introducedat day 28, and water is again introduced at day 35. δTE-13′supplementation continues throughout the study. The δTE-13′supplementation at 0.025% diet had no effect on body weight and foodintake (data not shown), but significantly attenuated DSS-inducedinflammation as indicated by decreased fecal bleeding during the secondcycle of DSS. δTE-13′ significantly suppressed AOM/DSS-inducedtumorigenesis in the colon, as indicated by decreased total tumormultiplicity by 34% (P<0.01) and large polyps by 58% (P<0.05). Theseresults indicate that δTE-13′-COOH appears to be safe and effective insuppression of colon cancer.

For example, in panel B of FIG. 5, the fecal bleeding score (0-3) isprovided for the 7^(th) day of the 2^(nd) DSS cycle. As shown in thepanel, the treatment group has a significantly decreased fecal bleedingscore compared to the control (p<0.05). Similarly, in panel C of FIG. 5,the total number of tumors and number of large (>2 mm²) tumors issignificantly decreased in mice receiving the δTE-13′ treatment comparedto a control. N=15-16.

Turning now to FIG. 6, using liquid chromatography-mass spectrometry,the formation of δTE-13′-1 (Scheme 6, the 3rd structure) in fecalsamples of mice fed with δTE-13′ was detected. Due to structuralsimilarity with other 13′ analogs, such as δTE-13′, we concluded thatthis compound contributes to anti-inflammatory and anticancer effects.

In FIG. 7, data from a DSS-induced colitis model indicates that δTE-13′in diet significantly mitigated DSS-induced diarrhea and fecal bleeding.In panel A, the study design is shown indicating that DSS and δTE-13′are introduced at day 0 and tissue is harvested at day 8 or 9. In panelB, the colitis score is determined for an untreated control (nocolitis), a DSS-treated control (high colitis score), and a DSS andδTE-13′-treated mouse (significantly decreased colitis score compared tothe DSS-treated control). These results indicate that δTE-13′ may beused to treat inflammatory bowel diseases including colitis.

Although carboxychromanols appear to be able to bind to the AA bindingsite and therefore can presumably inhibit COX activity by competing withthe substrate binding, the exact mechanism underlying the inhibitionneeds to be further elucidated. COXs are bifunctional enzymes that carryout two sequential activities, i.e., the cyclooxygenase activity whichleads to the formation of prostaglandin G₂ (PGG₂) and peroxidaseactivity which reduces PGG₂ to PGH₂ (28). Inhibition of peroxidaseactivity does not require specific binding to the AA site. In theory,chromanol analogs are able to inhibit peroxidase activity, like otherphenolic reductants. In fact, O'Leary et al. (29) reported that γ-T andα-T inhibit peroxidase activity of COX-2. However, it is believed thatthere is no direct correlation between the efficacy as a peroxidasereductant and its potency as an inhibitor of the COX activity (28). Ourcurrent and five previous studies (13) indicate that vitamin E forms areweak inhibitors of COXs.

The inhibitory effect of 9′, γ-CEHC, and certain forms of vitamin Eshowed discrepancy between cell-culture and enzyme-based assays. Thus,in IL-1β activated A549 cells, γ-T, δ-T and γ-TE reduced PGE₂ formation,even in the presence of sesamin which blocks carboxychromanol formation(FIGS. 1 and 2). 9′ and γ-CEHC inhibited COX-2 activity in intact cellswhere COX-2 was pre-induced and exogenous AA was added. In contrast,these compounds are less effective in enzyme-based assays (Table 1).This selectivity between cellular and enzyme studies resembles scenariosof weak COX inhibitors, e.g. acetaminophen and salicylate, which havebeen reported to inhibit COX activity in certain cellular environmentsbut are largely in vain in assays with purified enzymes (20, 30, 31).The observed selectivity has been attributed to the difference in lipidhydroperoxide generation (30, 31). Compared with cultured cells wherethe formation of PGG₂ is moderate because of limited induction of theenzyme and AA release, PGG₂ is often generated in much higher quantitiesin assays using purified enzyme (30, 31). Consistently, addition oflipid peroxide, e.g. PGG₂, antagonizes inhibitory effect ofacetaminophen and salicylate (30, 31). Based on the current study, weconclude that like acetaminophen and salicylate, γ-CEHC and vitamin Eforms are weak COX inhibitors, and they may inhibit COX activity onlywhen lipid hydroperoxide is relatively low, e.g. low levels of COX andAA. 9′ is also less efficient in enzyme-based assays (Table 1), but itsIC50 needs to be further determined.

One important implication of our current findings is that differentbioactivity among vitamin E forms may be rooted in their distinctmetabolism. To this end, long-chain carboxychromanols may contribute toin vivo anti-inflammatory effect of γ-T (3-2). We and others havedemonstrated that γ-T inhibited proinflammatory eicosanoids at theinflammatory site and attenuate inflammation-caused damage in variousanimal models (14, 33-35). Himmelfard et al. reported that γ-T enrichedbut not α-T-enriched mixed tocopherol inhibited C-reactive protein andIL-6 in kidney-dialysis patients 36). We recently showed thatsignificant amounts of 13′ but not 9′ were detected in the plasma andliver after γ-T supplementation (17), although pharmacokinetics of 13′formation needs to be further established. Our preliminary data showedthat relatively large amounts of 13′ (>100 nmol/g) were found in fecesas a result of γ-T supplementation in rats. This suggests that 13′, apotent inhibitor of COX and potentially abundant in colon tissues, couldalso contribute to the anti-cancer effect of mixed tocopherols enrichedwith γ-T and δ-T on ACF in AOM-induced colon cancer in rodent (Newmark,2006).

In addition, long-chain carboxychromanols and their analogs may beuseful and novel anti-inflammatory agents. We found that besidesinhibition of COX-1 and COX-2, 9′ and 13′ appeared to also inhibit5-lipoxygenase activity, which is a key enzyme to catalyze generation ofpro-inflammatory leukotrienes. Targeting on both COX and lipoxygenase isparticularly interesting because inhibition of these multiple pathwayscan not only result in more potent anti-inflammatory effect, but alsoreduce potential adverse effect caused by a shunt in arachidonatemetabolism to either pathway.

We found that 13′-carboxychromanol (Scheme 4), a long-chaincarboxychromanol which is derived from vitamin E, inhibited COX-2 andCOX-1 activity with IC50 at low microM (4-7 μM) concentrations, as shownin COX activity assays in intact cells and using purified COX-1 andCOX-2. The inhibitory potency is similar to ibuprofen (IC50-5 μM).

Although another metabolite, 9′-COOH, also inhibited COX-2 activity inassays using intact cells, but at up to 20 μM, it did not inhibit COX-1or COX-2 in the assays using purified COX-1 or COX-2, which indicatesthat 9′-COOH is a much weaker inhibitor than 13′-COOH. These studiesalso indicate that the inhibitory potency depends on the length of theside chain (consistently, 3′-COOH is a weaker inhibitor, with anIC50>3000/1 in the enzyme assay).

13′-COOH and 9′-COOH inhibited 5-LOX activity as assayed in HL-60 cellsdifferentiated neutrophils (estimated IC50 is at low microMconcentrations).

We previously found that 3′-carboxychromanol inhibited PGE₂ and LTB₄ atthe site of inflammation in a rat's inflammation model (62). Togetherwith the data described above, 13′-carboxychromanol and/or otherlong-chain carboxychromanols can be much more potent than3′-carboxychromanol in vivo.

In addition, carboxychromanols are potent antioxidants which may haveeffect on gene expression of cytokine expression such as TNFα (62).

Taken together, long-chain carboxychromanols are likely usefulanti-inflammatory agents because these compounds target multi-pathwayswhich are important to regulation of inflammatory response.

Because sesamin appears to inhibit β-oxidation which metabolizeslong-chain carboxychromanols, the addition of sesamin withcarboxychromanols will prolong the half life of carboxychromanols, andtherefore enhance the effect. Polyphenolic compounds are known toinhibit sulfotransferase activity, which leads to inhibition ofsulfation. Our preliminary data indicated that that polyphenols such ascurcumin inhibits sulfation of carboxychromanols (sulfatedcarboxychromanols do not appear to inhibit COX activity). Thecombination of polyphenols with long-chain carboxychromanols is likelyto enhance the efficiency.

As cyclooxygenases and lipoxygenases contribute to cancer development,long-chain carboxychromanols, and their analogs, or their combinationswith other bioactive compounds such as sesamin or polyphenoliccompounds, are likely to be effective cancer prevention and therapeuticagents. Because chronic inflammation has been implicated in otherchronic diseases including cardiovascular diseases, and age-relatedneurodegenerative diseases, carboxychromanols can be used as therapeuticagents against these diseases.

The description of the present disclosure has been presented forpurposes of illustration and description, but is not intended to beexhaustive or limited to embodiments of the disclosure in the formdisclosed. Many modifications and variations will be apparent to thoseof ordinary skill in the art without departing from the scope and spiritof embodiments of the disclosure. The embodiment was chosen anddescribed in order to best explain the principles of embodiments of thedisclosure and the practical application, and to enable others ofordinary skill in the art to understand embodiments of the disclosurefor various embodiments with various modifications as are suited to theparticular use contemplated. Although specific embodiments have beenillustrated and described herein, those of ordinary skill in the artappreciate that any arrangement which is calculated to achieve the samepurpose may be substituted for the specific embodiments shown and thatembodiments of the disclosure have other applications in otherenvironments. This application is intended to cover any adaptations orvariations of the present disclosure. The following claims are in no wayintended to limit the scope of embodiments of the disclosure to thespecific embodiments described herein.

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1. A pharmaceutical formulation comprising a conjugated13′-carboxychromanol compound selected from the group consisting of:

wherein R is selected from the group consisting of —H, —CH₃, and—(CO)CH₃, or a pharmaceutically acceptable salt thereof.
 2. Thepharmaceutical formulation of claim 1, wherein said compound is of theformula:


3. The pharmaceutical formulation of claim 1, wherein said compound isof the formula:


4. The pharmaceutical formulation of claim 1, wherein said compound isof the formula:


5. The pharmaceutical formulation of claim 1, further comprising apharmaceutically acceptable polyphenolic sulfation inhibitor.
 6. Thepharmaceutical formulation of claim 5, wherein said pharmaceuticallyacceptable polyphenolic sulfation inhibitor is sesamin or curcumin.
 7. Amethod for treating a physiological disorder associated withinflammation in the gut, the method comprising administering atherapeutic amount of a conjugated 13′-carboxychromanol compoundselected from the group consisting of:

wherein R is selected from the group consisting of —H, —CH₃, and—(CO)CH₃, or a pharmaceutically acceptable salt thereof.
 8. The methodof claim 7, wherein said compound is of the formula:


9. The method of claim 7, wherein said compound is of the formula:


10. The method of claim 7, wherein said compound is of the formula:


11. The method of claim 7, wherein said physiological disorder isselected from the group consisting of inflammatory bowel disease,gastritis, irritable bowel syndrome, ulcerative colitis, Crohn'sdisease, and diarrhea.
 12. The method of claim 7, wherein said patientis a human.
 13. The method of claim 7, wherein said patient isadditionally administered a pharmaceutically acceptable polyphenolicsulfation inhibitor.
 14. The method of claim 13, wherein saidpolyphenolic sulfation inhibitor is sesamin or curcumin.
 15. A methodfor treating cancer, the method comprising administering a therapeuticamount of a conjugated 13′-carboxychromanol compound selected from thegroup consisting of:

wherein R is selected from the group consisting of —H, —CH₃, and—(CO)CH₃, or a pharmaceutically acceptable salt thereof.
 16. The methodof claim 15, wherein said compound is of the formula:


17. The method of claim 15, wherein said compound is of the formula:


18. The method of claim 15, wherein said patient is additionallyadministered a pharmaceutically acceptable polyphenolic sulfationinhibitor.
 19. The method of claim 15, wherein the pharmaceuticallyacceptable polyphenolic sulfation inhibitor is selected from the groupconsisting of sesamin or curcumin.
 20. The method of claim 15, furthercomprising inhibiting at least one of COX-1, COX-2, and 5-LOX activity.